Continuous Exposure to Low-Dose Cisplatin and Apoptosis

Abstract

Cisplatin (CDDP) is widely used for the treatment of a variety of human malignancies.1) CDDP is a well-known DNAdamaging agent, and it is currently thought that DNA platination is an essential first step in its cytotoxic activity. [2][3][4][5] Recent evidence has shown that apoptosis is a marker of tumor cells that have been exposed to CDDP. [6][7][8] In addition, CDDP has been reported to induce apoptosis at higher concentrations than the clinical dose.The CDDP is a type of drug whose effects depends on the area under the concentration-time curve (AUC ), 9,10) which means that continuous exposure low concentrations of the drug have the same effect as a single short-term exposure at a high concentration. Continuous infusion or multiple administrations of low-dose CDDP is an excellent regimen for cancer patients. 11,12) For this reason, sustained release formulations of CDDP or CDDP derivatives have been developed. [13][14][15][16] However, it has not been clear whether continuous exposure with low-dose CDDP can induce apoptosis as effectively as a single high-dose exposure CDDP.We therefore compared the ability of CDDP to induce apoptosis when delivered as a single high-dose exposure or as multiple low-dose exposures. In these studies, the induction of apoptosis was assessed by measuring caspase-3 activity, DNA fragmentation, and the percent of cells in the sub-G1 phase. MATERIALS AND METHODSCell Culture Rat ascites hepatoma AH-109A cells were serially transplanted by intraperitoneal injection in Donryu rats. The cells taken from ascites were grown in RPMI-1640 medium containing 20% fetal bovine serum.Drug Exposure Cells were seeded into 25 cm 2 flasks at 4ϫ10 5 cells/ml. Single exposures to CDDP (1.0 mg/ml, Kyowa Hakko Kogyo, Tokyo, Japan) were carried out at the starting point (0 h). When CDDP was delivered as 8 low-concentration exposures, the cells were dosed every 2 h between 0 to 9 h and between 24 to 33 h, and when 16 low-concentration doses, it was delivered every 1.5 h between 0 and 10.5 h and between 24 to 34.5 h.Growth Inhibition Assay Cells were seeded into 96-well plates at 4ϫ10 5 cells/0.1 ml/well following single or multiple exposures to CDDP. To determine the growth inhibitory effect, 20 ml of a solution containing 2 mM WST-1 (Dojindo, Kumamoto, Japan) and 0.2 mM 1-methoxy methylphenazinium methylsulfate was added to each well followed by incubation for 6 h. The absorbance of each well was measured at 450 nm with a reference wavelength of 650 nm using a Model 550 microplate reader (BIO-RAD, Tokyo, Japan).DNA Fragmentation Assay DNA laddering in apoptotic cells was detected using an ApopLadder Ex kit (Takara Bio, Shiga, Japan). Briefly, cells were suspended in lysis buffer and centrifuged. The supernatants were mixed with 10% Sodium dodecylsulfate (SDS) and Enzyme A and then incubated at 56°C for 1 h. Enzyme B was then added, and the mixture was incubated for an additional 1 h at 37°C. To precipitate DNA fragments, the preparation was mixed with precipitant and ethanol and then stored for 15 min a...

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